Handbook of Fluorescence Spectroscopy and Imaging

From Ensemble to Single Molecules

Markus Sauer, Johan Hofkens, Jörg Enderlein

• Publisher: Wiley-VCH
• Year: 2010
• Print ISBN:
  9783527316694
• eBook ISBN:
  9783527633524
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Spectroscopy & Spectrum Analysis

Providing much-needed information on fluorescence spectroscopy and microscopy, this ready reference covers detection techniques, data registration, and the use of spectroscopic tools, as well as new techniques for improving the resolution of optical microscopy below the resolution gap.
Starting with the basic principles, the book goes on to treat fluorophores and labeling, single-molecule fluorescence spectroscopy and enzymatics, as well as excited state energy transfer, and super-resolution fluorescence imaging.
Examples show how each technique can help in obtaining detailed and refined information from individual molecular systems.

Chapter 1

Basic Principles of Fluorescence Spectroscopy

1.1 Absorption and Emission of Light

As fluorophores play the central role in fluorescence spectroscopy and imaging we will start with an investigation of their manifold interactions with light. A fluorophore is a component that causes a molecule to absorb energy of a specific wavelength and then re-remit energy at a different but equally specific wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. Fluorophores are also denoted as chromophores, historically speaking the part or moiety of a molecule responsible for its color. In addition, the denotation chromophore implies that the molecule absorbs light while fluorophore means that the molecule, likewise,emits light. The umbrella term used in light emission is luminescence, whereas fluorescence denotes allowed transitions with a lifetime in the nanosecond range from higher to lower excited singlet states of molecules.

In the following we will try to understand why some compounds are colored and others are not. Therefore, we will take a closer look at the relationship of conjugation to color with fluorescence emission, and investigate the absorption of light at different wavelengths in and near the visible part of the spectrum of various compounds. For example, organic compounds (i.e., hydrocarbons and derivatives) without double or triple bonds absorb light at wavelengths below 160 nm, corresponding to a photon energy of >180 kcal mol⁻¹ (1 cal = 4.184 J), or >7.8 eV (Figure 1.1), that is, significantly higher than the dissociation energy of common carbon-to-carbon single bonds.

Figure 1.1 The electromagnetic spectrum.

Below a wavelength of 200 nm the energy of a single photon is sufficient to ionize molecules. Therefore, photochemical decomposition is most likely to occur when unsaturated compounds, where all bonds are formed by σ-electrons, are irradiated with photon energies >6.2 eV. Double and triple bonds also use π-electrons in addition to a σ-bond for bonding. In contrast to σ-electrons, which are characterized by the rotational symmetry of their wavefunction with respect to the bond direction, π-electrons are characterized by a wavefunction having a node at the nucleus and rotational symmetry along a line through the nucleus. π-bonds are usually weaker than σ-bonds because their (negatively charged) electron density is further from the positive charge of the nucleus, which requires more energy. From the perspective of quantum mechanics, this bond weakness is explained by significantly less overlap between the component π-orbitals due to their parallel orientation. These less strongly bound electrons can be excited by photons with lower energy. If two double bonds are separated by a single bond, the double bonds are termed conjugated. Conjugation of double bonds further induces a red-shift in the absorption (a so-called bathochromic shift). All fluorophores that have a high absorption in the visible part of the spectrum possess several conjugated double bonds.

Above 200 nm only the two lowest energy transitions, that is, n → π∗ and π → π∗, are achieved as a result of the energy available from the photons. When sample molecules are exposed to light having an energy that matches a possible electronic transition within the molecule, some of the light energy will be absorbed as the electron is promoted to a higher energy orbital. As a simple rule, energetically favored electron promotion will be from the highest occupied molecular orbital (HOMO), usually the singlet ground state, S₀, to the lowest unoccupied molecular orbital (LUMO), and the resulting species is called the singlet excited stateS₁. Absorption bands in the visible region of the spectrum correspond to transitions from the ground state of a molecule to an excited state that is 40–80 kcal mol⁻¹ above the ground state. As mentioned previously, in saturated hydrocarbons in particular, the lowest electronic states are more than 80 kcal mol⁻¹ above the ground state, and therefore they do not absorb light in the visible region of spectrum. Such substances are not colored. Compounds that absorb in the visible region of the spectrum (these compounds have color) generally have some weakly bound or delocalized electrons. In these systems, the energy difference between the lowest LUMO and the HOMO corresponds to the energies of quanta in the visible region.

On the other side of the electromagnetic spectrum, there is a natural limit to long-wavelength absorption and emission of fluorophores, which is in the region of 1 µm [1]. A dye absorbing in the near-infrared (>700 nm) has a low-lying excited singlet state and even slightly lower than that, a metastable triplet state, that is, a state with two unpaired electrons that exhibits biradical character. Even though no generally valid rule can be formulated predicting the thermal and photochemical stability of fluorophores, the occupation of low-lying excited singlet and triplet states potentially increases the reactivity of fluorophores. Therefore, it is likely that fluorophores with long-wavelength absorption and emission will show less thermal and photochemical stability, due to reactions with solvent molecules such as dissolved oxygen, impurities, and other fluorophores. In addition, with increasing absorption, that is, with a decreasing energy difference between S₁ and S₀, the fluorescence intensity of fluorophores decreases owing to increased internal conversion. That is, with a decreasing energy difference between the excited and ground state, the number of options to get rid of the excited-state energy by radiationless deactivation increases. Hence, most known stable and bright fluorophores absorb and emit in the wavelength range between 300 and 700 nm.

Fluorophores with conjugated doubled bonds (polymethine dyes) are essentially planar, with all atoms of the conjugated chain lying in a common plane linked by σ-bonds. π-electrons, on the other hand, have a node in the plane of the molecule and form a charge cloud above and below this plane along the conjugated chain (Figure 1.2). The visible bands for polymethine dyes arise from electronic transitions involving the π-electrons along the polymethine chain. The wavelength of these bands depends on the spacing of the electronic levels. The absorption of light by fluorophores such as polymethine dyes can be understood semiquantitatively by applying the free-electron model proposed by Kuhn [2, 3]. The arrangement of alternating single–double bonds in an organic molecule usually implies that the π-electrons are delocalized over the framework of the “conjugated” system. As these π-electrons are mobile throughout the carbon atom skeleton containing the alternating double bonds, a very simple theoretical model can be applied to such a system in order to account for the energy of these electrons in the molecule. If one makes the seemingly drastic assumption that the several π-electrons that comprise the system are non-interacting (presumably, if the π-electrons are delocalized over the

C

C

C

C

C

C

framework, they spread out, minimizing repulsion between them), then one can view the energetics of this system as arising from the simple quantum mechanical assembly of one-electron energy levels appropriate to the particle in the box model. In this case, one considers the potential energy of the electron as being constant throughout the length of the molecular box and then rising to infinity at each end of the conjugated portion of the molecule. As an example, consider a positively charged simple cyanine dye. The cation can “resonate” between the two limiting structures shown in Figure 1.2 a, that is, the wavefunction for the ion has equal contributions from both states. Thus, all the bonds along this chain can be considered equivalent, with a bond order of 1.5, similar to the C

C bonds in benzene.

Figure 1.2 (a) Limiting structures of a resonance hybrid of a simple positively charged cyanine dye. (b) The π-electron cloud of the cyanine dye as seen from the side in a simplified potential energy (V) trough of length L.

Assuming that the conjugated chain extends approximately one bond length to the left and right beyond the terminal nitrogen atoms, application of the Schroedinger equation...

Table of contents

• Cover
• Title Page
• Copyright
• Preface
• Chapter 1: Basic Principles of Fluorescence Spectroscopy
• Chapter 2: Fluorophores and Fluorescent Labels
• Chapter 3: Fluorophore Labeling for Single-Molecule Fluorescence Spectroscopy (SMFS)
• Chapter 4: Fluorophore Selection for Single-Molecule Fluorescence Spectroscopy (SMFS) and Photobleaching Pathways
• Chapter 5: Fluorescence Correlation Spectroscopy
• Chapter 6: Excited State Energy Transfer
• Chapter 7: Photoinduced Electron Transfer (PET) Reactions
• Chapter 8: Super-Resolution Fluorescence Imaging
• Chapter 9: Single-Molecule Enzymatics
• Index